The proposed study will give information about the localization, binding and mobility of cholestrol in the erythrocyte membrane. Cholesterol can be removed from or added to the red cell membrane, while leaving the membrane composition unaltered in all other respects, by incubating the cells with phospholipid-cholesterol liposomes containing varying amounts of cholesterol. Experiments will be carried out to determine whether the cholesterol initially removed from the membrane when red cells are incubated with phospholipid liposomes come from an environment which is different from that of the rest of the membrane cholesterol. Radioactively labelled cholesterol of know specific activity will be introduced into cholesterol depleted red cells. After a period of time which will be varied in the experiment, cholesterol again will be removed from the cells by incubating them wit liposomes. An analysis of the difference between the special activity of the cholesterol introduced into the membrane and that subsequently removed from it will give information about the number and types of environments of cholesterol in the red cell membrane. Abnormal erythrocytes containing excess cholersterol, such as the spur cells found in patientss with liver disease also will be studied by this method to determine whether the excess cholersterol has a distinguishable environment and hence is segregated from the rest of the membrane cholesterol. The exchange of cholesterol between normal red cells metabolically labelled with 3H-cholesterol in vivo and liposomes will be studied to determine how many cholerterol pools there are in the membrane and the rate of exchange between pools if more than one is found. These experiments will also measure the rte of exchange of cholesterol ("flip-flop") between the two halves of the bilayer in phospholipid-cholesterol liposomes. The characteristics of cholesterol exchange and removal from the inner and outer surface of the erythrocyte membrane will be studied using membrane vesicles of inside-out and right-side-out orientation. Normal and cholesterol-rich erythrocytes will be studied by autoradiography to obtain information about the distribution of cholesterol in the plane of the membrane in these cells.